Ghrelin blunted vascular calcification in vivo and in vitro in rats

Regulatory Peptides 129 (2005) 167 – 176 www.elsevier.com/locate/regpep

Ghrelin blunted vascular calcification in vivo and in vitro in rats Gui-zhong Lia,b, Wei Jiangc,d, Jing Zhaoc, Chun-shui Pana,d, Jun Caob, Chao-shu Tanga,c,d, Lin Changa,c,d,T a

Institute of Cardiovascular Disease Research, the First Hospital of Peking University, Beijing 100034, PR China b Department of Pathophysiology, Ningxia Medical College, Yingchuan 750001, PR China c Department of Physiology, Peking University Health Science Center, Beijing 100083, PR China d The Key Lab of Cardiovascular Molecular Biology, Ministry of Education, Beijing 100083, PR China Received 20 October 2004; accepted 4 February 2005 Available online 22 March 2005

Abstract Ghrelin is a new peptide with regulatory actions in growth hormone secretion in the anterior pituitary gland and in energy metabolism. Currently, ghrelin has potently protective effects in cardiovascular diseases. We used an in vivo model of rat vascular calcification induced by vitamin D3 and nicotine and one of cultured rat vascular smooth muscular cells (VSMCs) calcification induced by h-glycerophosphate to study the possible mechanism in the regulatory action of ghrelin in vascular calcification. Calcification increased total Ca2+ content and 45 Ca2+ deposition in aortas and VSMCs and alkaline phosphatase (ALP) activation in plasma, aortas and VSMCs. However, calcified aortas and VSMCs showed a significant decrease in osteopontin (OPN) mRNA expression and a marked reduction of ghrelin levels in plasma and its mRNA expression in aortas. The aortic calcification was significantly attenuated by subcutaneous administration of ghrelin 30 and 300 nmol kg 1 day 1 for 4 weeks, and the latter dosage was more potent than the former. Ghrelin treatment at the two dosages reduced the total aorta Ca2+ content by 24.4% and 28.1%, aortic 45Ca2+ deposition by 18.4% and 24.9%, plasma ALP activity by 36.6% and 76.7%, and aortic ALP activity by 10.3% and 47.6% (all P b 0.01 or 0.05), respectively. Ghrelin at 10 8–10 6 mol/L attenuated the calcification in cultured VSMCs, with decreased total Ca2+ content, 45Ca2+ deposition and ALP activity and increased OPN mRNA expression, in a concentrationdependent manner. In addition, endothelin levels in plasma and aortas and its mRNA expression in aortas significantly increased with calcification, but ghrelin treatment significantly decreased endothelin levels and mRNA expression, with the high dosage being more potent than the lower dosage. These results indicate that local ghrelin in vascular was down-regulated during vascular calcification, whereas administration of ghrelin effectively attenuated vascular and VSMCs calcification. D 2005 Elsevier B.V. All rights reserved. Keywords: Vascular; Calcification; Ghrelin; Rat

1. Introduction Vascular calcification is associated with pathological processes such as atherosclerotic lesions, diabetes mellitus, vascular lesions in the nephritic syndrome, vascular endothelial injury and aging [1]. Although previously considered uncommon, vascular calcification is now known

T Corresponding author. Institute of Cardiovascular Disease Research, The First Hospital of Peking University, Beijing 100034, PR China. Tel.: +86 10 82802851; fax: +86 10 66551036. E-mail address: [email protected] (L. Chang). 0167-0115/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2005.02.015

to be present in 80% of significant lesions and in at least 90% of patients with coronary artery disease [2]. Vascular calcification is an important risk factor for cardiovascular events because it causes decreased aortic compliance and elastic recoil, which, in severe cases, results in cardiac ischemia due to significantly impaired reverse aortic flow and coronary perfusion [1]. It was long believed to be an end-stage process of bpassiveQ mineral precipitation. However, there is now a growing awareness that vascular calcification is a biologically regulated phenomenon similar to bone formation and osteoporosis [2,3]. Currently, no effective treatment strategy protected heart and vessels against calcification, because little is known about its exact

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pathogenetic mechanism [1]. Recently, a few cytokines (e.g., interleukin-6), growth factors (e.g., platelet derived growth factor) and vascular active peptides (such as adrenomedullin, angiotensin II, endothelin, parathyroid hormone-relative peptide and C-type natriuretic peptide), which are related to maintain cardiovascular function and structure, have been found to be involved in the regulation of vascular calcification [3]. The use of endogenous inhibitory factors may point to a new therapeutic strategy for regulating vascular calcification [4]. Ghrelin is a newly discovered peptide originating mainly from gastric mucosa. It is a 28 amino acid acyl peptide esterified with octanoic acid on Ser 3 and belongs to the brain–gut peptide family [5]. Endocrine activities of ghrelin are mediated by growth hormone (GH) secretagogue receptor (GHSR)-1a, a G-protein-coupled receptor mainly expressed in the pituitary gland and hypothalamus, identified previously as the receptor for GH secretagogues (GHS), through which ghrelin induces GH release and stimulates food intake and adiposity [6]. Recent evidence indicates that ghrelin and synthetic GHS play various cardiovascular activities. They are involved in increasing myocardial contractility, vasodilatation, antagonizing the vascular effects of endothelin-1 [7] and protecting against myocardial infarction-induced heart failure or dysfunction of vascular endothelium in vivo [8], as well as attenuating injury in the heart from ischemia/reperfusion in vitro [9]. In addition, since the ghrelin receptor, GHSR-1a, and the ghrelin mRNA are expressed in the myocardium and VSMCs [10], ghrelin may directly regulate the function and structure of the cardiovascular system as a local factor [11]. However, whether ghrelin also protects against vascular calcification has not been investigated. In this study, we established a rat model of vascular calcification with vitamin D3 and nicotine administration and cultured calcified VSMCs with h-glycerophosphate to observe the impact of ghrelin on vascular calcification and study its mechanisms.

2. Material and methods 2.1. Materials Male Sprague–Dawley rats (weighing 200–250 g) were purchased from the animal center, Health Sciences Center of Peking University, and were housed under standard conditions (room temperature 20 F 1 8C, humidity 60 F 10%, lights from 6 a.m. to 6 p.m.) and given standard rodent chow and water freely. All experimental procedures were performed in accordance with the Guidelines of Animal Experiments from the Committee of Medical Ethics, National Health Department of China (1998). Vitamin D3, nicotine, h-glycerophosphate and an alkaline phosphatase assay kit were obtained from Sigma (St. Louis, MO, USA); radioimmunoassay kits for ghrelin and endothe-

lin were kindly provided by Phoenix Pharmaceutical (Belmont, CA, USA); 45CaCl2 was from NEN Life Sci (Boston, MA, USA); Dulbecco’s modified Eagle medium (DMEM) and Trizol from GIBCO BRL (Gaithersburg, MD, USA); dNTP from Clontech Laboratories (Palo Alto, CA, USA); and Moloney murine leukemia virustranscriptase (MMLV), Taq, RNAsin and Oligo(dT)15 primer were from Promega (Madison, WI, USA). Oligonucleotides were synthesized by Sai Baisheng Biotechnology (Beijing, China). The sequences of oligonucleotide primers were as follows: ghrelin-S, 5V-TTGAGCCCAGAGCACCAGAAA-3V; ghrelin-A, 5V-AGTTGCAGAGGAGGCAGAAGCT-3V; osteopontin (OPN)-S, 5V-CTCGCGGTGAAAGTGGCTGA-3V; OPN-A, 3V-GACCTCAGAAGATGAACTCT-5V; endothelin-S, 5V-GGTCTTGATGCTGTTGCTGA-3V; endothelin-A, 5V-GAGCTGAGAAGGAAGTGCAGA-3V, as well as hactin-S, 5V-ATCTGGCACCACACCTTC-3V; and h-actin-A, 5V-AGCCAGGTCCAGACGCA-3V for calibration of sample loading. Other chemicals and reagents were of analytical grade. 2.2. Preparation of vascular calcification model in vivo in rats Rats were randomly divided into the following groups (6 rats in each group): control group, calcified group (VDN group) and calcified group treated with ghrelin 30 nmol kg 1 day 1 (VDN + Ghr[L] group) or 300 nmol kg 1 day 1 (VDN + Ghr[H] group). The preparation for vascular calcification was as previously described [12]. In short, the rats in VDN group were intramuscularly treated with 300,000 IU/kg vitamin D3 and nicotine (25 mg/kg in olive oil, given orally), and nicotine was given again after 8 h. The VDN + Ghr groups were treated with the same dosages of vitamin D3 and nicotine at the same time, then subcutaneously treated with 30 or 300 nmol kg 1 day 1 ghrelin for 4 weeks. The control group followed a similar protocol, except that animals received normal saline intramuscularly and two gavages of olive oil (5 mL/kg orally). After blood pressure and heart rate were measured in conscious animals by placing a cuff connected to a sphygmomanometer (Model RBP-1) on the base of the tail, the rats were anesthetized with 30 mg/kg pentobarbital sodium. Blood were collected and the thoracoabdominal aorta was removed to ice-cold phosphate buffered saline (PBS), then weighed and stored at 70 8C. 2.3. Cell culture and VSMC calcification Rat VSMCs were acquired by an explant method described by Campbell and Campbell [13]. Briefly, the tunica media was isolated from the rat aortas. The tissue was fragmented (1–2 mm3), placed in a 10-cm culture dish, and cultured for several weeks in DMEM containing 4.5 g/L of glucose supplemented with 15% fetal bovine serum (FBS) and 10 mM sodium pyruvate at 37 8C in a humidified

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atmosphere containing 5% CO2. Cells that had migrated from the explants were collected and maintained in the growing medium. Immunocytochemical examination showed positive staining in all cells for a´-smooth muscle actin. VSMCs calcification was induced as previously described [14]. In short, after VSMCs becoming confluent, the cells were seeded in DMEM containing 15% FBS and 10 mM sodium pyruvate in the presence of 10 mM hglycerophosphate (calcifying medium). The calcifying medium was refreshed every 2 days. In the time–course experiments, the beginning day of culture in calcifying medium was defined as day 0. Cells were grown in media as follows: 1) the control group received normal medium; 2) the calcifying group calcifying medium; 3) the ghrelin-alone group 10 6 mol/L ghrelin in normal medium; and 4) the ghrelin-treated group calcifying medium containing 10 8, 10 7 and 10 6 mol/L ghrelin. After 10 days of incubation, the following measurements were performed. 2.4. Determination for calcification The vascular and cellular calcification was assessed by uptake 45Ca and quantification of cellular or aortic calcium deposition. 45Ca accumulation in the VSMC layer was assessed as described in Ref. [14], after exposing cells to 37 kBq/mL 45CaCl2 for 24 h. Aortas (about 20 mg) [15] were sliced and incubated in 1 mL Krebs–Henseleit (K–H) solution (118 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, and 11 mM glucose) with 37 kBq/mL of 45CaCl2 for 12 h. The reaction was stopped by the addition of ice-cold K–H solution. The aortic tissue was dissolved in foric acid, and protein content was determined by Bradford’s method. 45 Ca2+ radioactivity in VSMCs and aortas was measured by scintillation counting (Beckman LS 6500). Quantification of cellular or aortic calcium deposition was performed according to Mori et al. [16], with some modifications. After the cell layer was incubated, the medium was removed and the layer was washed five times with cold PBS. Scraped cells were collected, dissolved in HNO3 and diluted with a blank solution (27 nmol/L KCl, 27 Amol/L LaCl3 in de-ionized water). Abdominal aortas [15] (10 mg) were directly dissolved in HNO3, then dried in an oven and re-dissolved with the blank solution. The calcium content was measured by use of an atomic absorption spectrophotometer at 422.7 nm. Calcium content of the cell layer and aorta was normalized by protein content. 2.5. Von Kossa staining for calcification Von Kossa calcium staining of cultured VSMCs was as described in Ref. [17]. Briefly, cell monolayers were fixed in 0.1% glutaraldehyde for 15 min at room temperature. Cells were then washed twice with ddH2O and incubated with 5% silver nitrate for 30 min at room temperature in the

169

dark. Silver nitrate was removed, and the cells were rinsed twice with ddH2O. After being air dried, cultures were exposed to sunlight until color development was complete. For calcium staining in aorta [15], 1 cm of the thoracic descending aorta just below the aortic arch was excised and immersed in 10% formol. Samples were dehydrated and embedded in paraffin. Six micron-thick sections were cut and stained with hematoxylin–eosin. The slides were deparaffinized and dehydrated before being immersed in a light-protected, silver nitrate solution (5 g in 100 mL distilled water) for 30 min under a 100-W bulb, then immersed in a solution of 5% sodium thiosulfate in distilled water for 2 min. Finally, slides were counterstained with safranine (red staining). 2.6. Assay for alkaline phosphatase (ALP) activity ALP activity in VSMCs and aortic tissue was assessed as described in Ref. [18], with the use of the ALP assay kit from Sigma. The cells were rinsed three times with cold PBS, scraped into 200 uL of lysis buffer (0.2% NP-40 in l mM MgCl2) and sonicated for 10 s. Next, l mL of reaction mixture (221 alkaline buffer:stock substrate solution 1:1, prepared by dissolving the contents of a 100-mg capsule of Sigma 104 phosphatase substrate in 25 mL of ddH2O) was added to each well. Homogenates of endothelium-denuded abdominal aortas (homogenized buffer: 20 mmol/L HEPES containing 0.2% NP-40 and 20 mmol/Lol/L MgCl2) was prepared by use of a Polytron. After centrifugation at 8,000  g for 10 min, the protein content of the supernatant was determined by Bradford’s method. The sample (200 Ag protein/200 uL) was mixed with l mL reaction mixture. The mixture was then incubated for 30 min at 37 8C. The yellow color indicated ALP activity. The reaction was stopped by the addition of 12 AL of 1 N NaOH to each well, and absorbance was determined at 405 nm. ALP activity was calculated with U-nitrophenol as the standard according to the manufacturer’s instructions. One unit was defined as the activity producing l nmol/L of U-nitrophenol for 30 min. 2.7. Determination of levels of immunoreactive ghrelin (irghrelin) and endothelin (ir-endothelin) in plasma and aortas Blood samples of each rat were taken with the use of ethylenediamine tetraacetic acid (EDTA)d Na2 (2.7 mM), aprotinin (500 KIU) and heparin. The plasma was separated by centrifugation (1,600  g for 15 min) and stored at 70 8C for assaying. Chopped aortic tissues were boiled for 10 min in l M acetic acid and homogenized, with a Polytron set at 4 8C. The extract solution was centrifuged at 24,000  g for 30 min. The plasma and tissue extract solution were loaded onto a Sep-Pak C18 cartridge (Waters, MA, USA) and pre-equilibrated with 0.5 mM acetic acid. The adsorbed material was eluted with 4 mL of 50% CH3CN containing 0.1% trifluoroacetic acid. After the samples were lyophi-

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lized, the residue was dissolved in radioimmunoassay buffer and assayed according to the manufacturer’s instructions. The IC50 of the ghrelin assay was 6.95 pg/tube and the reactivity with rat and human ghrelin 100%. No crossreactivity was found with human secretin, rat prolactin– releasin peptide-31 and rat gakanin. The IC50 of the endothelin assay was 14.6 pg/tube and the reactivity with rat and human endothelin 100%. No cross-reactivity was found with human angiotensin I, rat brain natriuretic peptide-31 and rat [Arg8]–vasopressin. The within-and between-assay coefficients of variation for both assay results were less than 10%. The content of ir-ghrelin and irendothelin was calculated and expressed as picomole per liter for plasma and picomole per gram wet weight for tissues, respectively. 2.8. Reverse-transcriptase polymerase chain reaction (RTPCR) assay The expression of OPN, ghrelin and endothelin mRNA was assessed by RT-PCR as described in Ref. [19]. Total RNA of aortic tissues or VSMCs was extracted from experimental groups with use of standard techniques. Isolated total tissue and cellular RNA were then quantified by use of an ultraviolet (UV) spectrophotometer (DU-640, Beckman, USA). Reverse transcription to cDNA was accomplished by priming 2 Ag of total RNA samples with MMLV and oligo (dT) 15 primer. The products were then used for the following PCR amplification: the PCR reaction mixture was in a 25-AL volume containing 2.5 mM dNTP 1 AL, 10  PCR buffer (20 mM MgCl2, 500 mM KCl, 1.5 M Tris–HCl, pH 8.7), 2.5 AL cDNA, 200 nM of the appropriate OPN, ghrelin- or endothelin-paired primers and 1.25 unit of Taq DNA polymerase. The following PCR cycles were used: 94 8C for 30 s, 57 8C for 30 s and 72 8C for 40 s, for 30 cycles, then 74 8C for 5 min. As an internal control for each PCR reaction, h-actin mRNA was also amplified with each sample. A total of 200 nM of actin primers and cDNA 2 AL was amplified under the same reaction conditions. All PCR products were loaded onto a 1.5% agarose–Tris–acetate–EDTA gel before electrophoresis, and products were visualized on ethidium bromide staining. The UV illumination photos then underwent computerized densitometric analysis. The final results are expressed as the ratios of OPN (871 bp), ghrelin (374 bp) or endothelin (446 bp) PCR product to the h-actin PCR product (291 bp) for each sample. All experiments were repeated three times. 2.9. Statistical analysis Results are shown as mean F SEM. Comparisons involved use of unpaired Student’s t-test and one-way ANOVA, followed by the Student–Newman–Keuls test. A P value b 0.05 was considered statistically significant.

3. Results 3.1. Calcium deposits of aorta and VSMCs Control aortas showed no calcified deposits (Fig. 1A), but calcified aortas showed strong positive staining among the elastic fibers of the medial layer (Fig. 1B). After treatment with ghrelin, the positive staining in the aortic medial layer was much less than that in the calcified aortas (Fig. 1C and D). As shown in Table 1, 4 weeks after vitamin D3 and nicotine treatment, the systolic pressure of the caudal artery and aortic calcium content in the VDN group was 23.3% and 11.3-fold higher, respectively (both P b 0.01), than that in controls. Compared with the VDN group, the ghrelin-treated groups (30 and 300 nmol kg 1 day 1) both showed decreased aortic calcium content, by 24.4% and 28.1%, respectively (both P b 0.01) but no change in systolic pressure (both P N 0.05). Compared with 30-nmol kg 1 day 1 ghrelin treatment, 300-nmol kg 1 day 1 treatment resulted in no significant difference in systolic pressure and aortic calcium content ( P N 0.05). The VDN group showed a 64.5% higher 45Ca2+ accumulation in aortas than that of control group ( P b 0.01). While treatment with ghrelin (30 and 300 nmol kg 1 day 1) decreased the 45Ca2+ accumulation by 18.4% and 24.9%, respectively (both P b 0.01). No significant difference was found in accumulation between the two ghrelin dosages ( P N 0.05). Control VSMCs showed no calcified deposits (no multicellular nodules) (Fig. 2A), but calcified VSMCs showed multicellular nodules (Fig. 2B). After co-incubated with ghrelin, the number of multicellular nodules decreased significantly (Fig. 2C). As shown in Table 2, compared with controls, VSMCs treated with h-glycerophosphate had a 4.4-fold higher calcium content ( P b 0.01). Treatment with 10 7 and 10 6 mol/L ghrelin, but not 10 8 mol/L, attenuated the h-glycerophosphateinduced calcification, with 32.4% and 33.3%, respectively (both P b 0.01), lower calcium content than that with hglycerophosphate alone. Incubation with ghrelin alone (10 6 mol/L) had no effect on cellular calcium content compared with controls ( P N 0.05). h-glycerophosphatetreated VSMCs had a 3.8-fold higher 45Ca2+ accumulation than control VSMCs ( P b 0.01). Attenuation of the hglycerophosphate-induced 45Ca2+ accumulation by 10 9, 10 8 and 10 7 mol/L ghrelin was concentration dependent, with 32.6–46.1% (all P b 0.01), respectively, lower accumulation than that with h-glycerophosphate alone. Incubation with ghrelin alone (10 6 mol/L) had no effect on cellular 45Ca2 accumulation ( P N 0.05). Compared with controls, VSMCs treated with h-glycerophosphate had a 3.8-fold higher 45Ca2+ accumulation ( P b 0.01). The attenuation of the h-glycerophosphate-induced accumulation by 10 9, 10 8 and 10 7 mol/L ghrelin was concentration dependent, with 32.6~46.1% (all P b 0.01), respectively, lower accumulation than that with h-glycerophosphate alone. However, incubation with ghrelin alone

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Fig. 1. Von Kossa staining for aortic calcification of multicellular nodules, showing a strong positive staining of black/brown areas (arrow shows) among the elastic fibers of the medial layer in calcified aorta (original magnification 40). A: controls; B: calcified aorta; C: calcified aorta treated with ghrelin 30 nmol kg 1 day 1 for 4 weeks; D: calcified aorta treated with ghrelin 300 nmol kg 1 day 1 for 4 weeks. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(10 6 mol/L) had no effect on 45Ca2+ accumulation in VSMCs compared with controls ( P N 0.05). 3.2. Ghrelin decreased ALP activity in plasma, aorta and VSMCs As shown in Table 1, compared with controls, the VDN group showed ALP activity in plasma and aortas that was 4.8- and 5.3-fold higher, respectively ( P b 0.01). Compared with the VDN group, those treated with ghrelin (30 and 300 nmol kg 1 day 1) both showed decreased ALP activity, by 36.6% and 76.7% in plasma (both P b 0.01), and 10.3% ( P N 0.05) and 53.0% ( P b 0.01) in aortas, respectively. Compared with 30-nmol ghrelin treatment, 300-nmol treatment resulted in 47.6% and 63.3% lower ALP activity in plasma and aortas, respectively (both P b 0.05).

As shown in Table 2, compared with controls, VSMCs treated with h-glycerophosphate had 6.0-fold higher ALP activity ( P b 0.01). Ghrelin (10 9, 10 8 and 10 7 mol/L) decreased the h-glycerophosphate-stimulated ALP activity in a concentration-dependent manner, with 12.1~38.6%, respectively (all P b 0.01), lower activity than that with hglycerophosphate treatment alone. However, incubation with ghrelin alone (10 6 mol/L) had no effect on ALP activity in VSMCs ( P N 0.05). 3.3. Ghrelin increased OPN mRNA expression in aortas and VSMCs As shown in Fig. 3, compared with controls, the VDN group showed 10.4% lower OPN mRNA expression in aorta ( P b 0.05). Groups treated with ghrelin (30 and 300 nmol)

Table 1 Mean weight, blood pressure (BP), calcium content, alkaline phosphatase (ALP) activity and Weight (g)

CON VDN VDN + Ghr(L) VDN + Ghr(H)

364 F 21 352 F 23 346 F 30 365 F 21

BP (mm Hg)

107 F 10 132 F 15** 126 F 10* 116 F 10

45

Ca2+ deposition in aortas and plasma of rats

Total aortic Ca2+ content (Amol/g [w/w])

Aortas (U/mg pro.)

Plasma (U/L)

Aortic 45Ca-deposition (Amol/g [w/w])

24.5 F 3.6 276.1 F 22.2** 208.6 F 16.1**# 198.4 F 32.5**#

69 F 12 368 F 122** 330 F 106** 173 F 53*#

36 F 7 172 F 10** 109 F 26**# 40 F 10*#

3.61 F 0.55 5.94 F 0.60** 4.85 F 0.93**# 4.46 F 0.4*#

ALP activity

Date were show as mean F SEM, n = 6 in each group. *P b 0.01 vs CON, #P b 0.01 vs VDN. Ghr indicates ghrelin; CON, controls; VDN, rats given vitamin D3 and nicotine for 4 weeks; VDN + Ghr(L), rats with vascular calcification treated with ghrelin 30 nmol kg 1 day 1 for 4 weeks; VDN + Ghr(H), rats with vascular calcification treated with ghrelin 300 nmol kg 1 day 1 for 4 weeks.

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Fig. 2. Formation of multicellular nodules (arrow shows) in culture phase contrast microscopy showing rat VSMCs (original magnification 20). A: controls; B: VSMCs treated with h-glycerophosphate 10 3 mol/L for 10 days; C: VSMCs treated with h-glycerophosphate 10 3 mol/L and ghrelin 10 6 mol/L for 10 days.

Table 2 Total Ca2+ content, ALP activity,

45

2+

Ca

deposition in rat VSMCs

Total Ca2+ content ALP activity (U/105 cells) (nmol/105 cells) CON 30.4 F 4.31 CAL 135.3 F 12.26* Ghr (10 6 mol/L) 31.4 F 4.90# CAL + Ghr 137.2 F 11.14* (10 8 mol/L) CAL + Ghr 91.5 F 9.93*,# (10 7 mol/L) CAL + Ghr 90.2 F 11.12*,# (10 6 mol/L)

91.8 F 7.07 554.5 F 20.5* 96.9 F 10.89# 487.4 F 34.0*,#

45

Ca2+ deposition (pmol/105 cells) 0. 37 F 0.10 1.41 F 0.06* 0.38 F 0.04# 0.95 F 0.05*,#

402.8 F 28.8*,# 0.87 F 0.03*,# 340.7 F 33.2*,# 0.76 F 0.08*,#

Date were show as mean F SEM, n = 6 in each group. *P b 0.01 vs CON, # P b 0.01 vs CAL. Ghr indicates ghrelin; CON, controls; CAL, VSMCs treated with 10 3 mol/L h-glycerophosphate for 10 days; Ghr (10 6 mol/ L), VSMCs treated with 10 6 mol/L ghrelin for 10 days; CAL + Ghr (10 8 mol/L), VSMCs treated with 10 3 mol/L h-glycerophosphate and 10 8 mol/L ghrelin for 10 days; CAL + Ghr (10 7 mol/L), VSMCs treated with 10 3 mol/L h-glycerophosphate and 10 7 mol/L ghrelin for 10 days; CAL + Ghr (10 6 mol/L), VSMCs treated with 10 3 mol/L h-glycerophosphate and 10 6 mol/L ghrelin for 10 days.

3.4. Ghrelin and endothelin levels in rat plasma and aorta As shown in Table 3, compared with controls, the VDN group showed a 21.8% lower ghrelin level in plasma ( P N 0.05). Compared with the VDN group, those treated with ghrelin (30 and 300 nmol) both showed increased ghrelin content in plasma, by 122.4% and 214.7% (both P b 0.01), respectively. Compared with the rats treated with 30 nmol ghrelin, those administered 300 nmol showed a 41.5% higher ghrelin content in plasma ( P b 0.01). Compared with controls, the VDN group showed a 102% and

Relative OPN mRAN level(% of β-actin)

showed up-regulated OPN mRNA expression, by 34.4% and 75.2%, respectively ( P b 0.01), than the VDN group, and the 300-nmol treatment was more potent than the 30nmol treatment ( P b 0.01). As shown in Fig. 4, h-glycerophosphate down-regulated the OPN mRNA expression in VSMCs by 13.0% ( P b 0.01). However, ghrelin treatment (10 8–10 6mol/L) up-regulated the expression significantly in a concentration-dependant manner, by 27.7–79.0% (all P b 0.01), respectively, than that with h-glycerophosphate treatment alone.

300

CON

VDN VDN+Ghr(L) VDN+Ghr(H)

OPN mRNA β-actin

** #

200

** # *

100

0 CON

VDN

VDN+Ghr(L) VDN+Ghr(H)

Fig. 3. RT-PCR results showing expression of osteopontin (OPN) mRNA in rat aortas. Ghr indicates ghrelin; CON: controls; VDN: rats given vitamin D3 and nicotine for 4 weeks; VDN + Ghr(L): calcified aortas treated with ghrelin 30 nmol kg 1 day 1 for 4 weeks; VDN + Ghr(H), calcified aortas treated with ghrelin 300 nmol kg 1 day 1 for 4 weeks. *P b 0.05, **P b 0.01 vs CON; #P b 0.01 vs VDN.

Relative OPN mRNA level(% of β-actin)

G. Li et al. / Regulatory Peptides 129 (2005) 167–176

173

OPN mRNA

250

β-actin

200

*#

150

*# *#

100

*

50

0

(10-3 mol/L)

GP Ghrelin (mol/L)

-

+ -

10

-6

+

+

+

10-8

10-7

10-6

Fig. 4. Expression of osteopontin (OPN) mRNA in rat VSMCs. VSMCs were treated with 10 3 mol/L h-glycerophosphate (GP) and various concentrations of ghrelin (from 10 8 to 10 6 mol/L) for 10 days. Column 1 was control, and column 2 was calcification VSMCs. *P b 0.01 vs control, #P b 0.01 vs calcification VSMCs.

4. Discussion Vascular calcification, including coronary and aortic calcification, is common and clinically significant in atherosclerosis, heart failure, patients under renal dialysis and aging Table 3 Ghrelin and endothelin (ET) levels in plasma of rats

CON VDN VDN + Ghr(L) VDN + Ghr(H)

Plasma Ghrelin (pmol/L)

Plasma ET (pmol/L)

Aortic ET (pmol/g [w/w])

198.5 F 26.1 155.2 F 17.1 345.1 F 62.5**,## 488.4 F 72.7**,##

2.84 + 0.46 5.74 F 0.47** 3.66 F 1.02## 3.42 F 0.93##

1.38 F 0.18 2.81 F 0.30** 2.46 F 0.21**,# 2.18 F 0.20**,##

Date were show as mean F SEM, n = 6 in each group. *P b 0.05, **P b 0.01 vs CON; #P b 0.05, ##P b 0.01 vs VDN. Ghr indicates ghrelin; CON, controls; VDN, rats given vitamin D3 and nicotine for 4 weeks; VDN + Ghr(L), rats with vascular calcification treated with ghrelin 30 nmol kg 1 day 1 for 4 weeks; VDN + Ghr(H), rats with vascular calcification treated with ghrelin 300 nmol kg 1 day 1 for 4 weeks.

[2]. Coronary calcification is present in most patients with coronary artery disease, in individual significant coronary artery lesions, and in ruptured lesions causing sudden death [1]. It is also the single most important risk factor in angioplasty [2]. Vascular calcification is usually associated with turbulence in vascular cells, including VSMCs, pericytes, and macrophages, which are transformed into an osteoblast-like phenotype and characterized by an increase in ALP activity, matrix vesicle formation and overexpression of bone morphogenetic proteins (BMPs), including BMP-2 and bone matrix proteins such as osteopontin, osteonectin, and osteocalcin [1]. Currently, vascular calcification has been recognized as a complex procession involving multiple factors [3]. Although some cardiovascular bioactive peptides have no direct action on calcium/phosphate regulation, they take part in the pathogenesis of vascular calcification as local factors with important regulatory paracrine/autocrine roles in vascular cells [2,3]. Other studies and our previous work indicated that local factors such as endothelin, angiotensin II Relative Ghrelin mRNA level (% of β-actin)

103% lower endothelin level in plasma and aorta, respectively (both P b 0.01). Compared with the VDN group, rat groups administered 30 or 300 nmol ghrelin showed decreased endothelin content in plasma and aortas, by 36.3% and 40.3% (both P b 0.01), respectively, but the contents in plasma and aortas were not significantly different between the groups (both P N 0.05). As shown in Fig. 5, compared with controls, the VDN group showed a 41.7% lower ghrelin mRNA expression in aortas ( P b 0.01), and a 28.4% higher endothelin mRNA expression ( P b 0.01; Fig. 6). However, ghrelin treatment (30 and 300 nmol) down-regulated the endothelin mRNA expression significantly, by 19.5% and 36.6%, respectively ( P b 0.01), the 300-nmol treatment being more potent than the 30-nmol treatment ( P b 0.01).

CON

VDN

Ghrelin mRNA β-actin

150 125 100

*

50 25 0 CON

VDN

Fig. 5. Expression of ghrelin mRNA in rat aortas. CON indicates controls; VDN, rats given vitamin D3 and nicotine for 4 weeks. *P b 0.01 vs CON.

Relative ET mRNA level (% of β-actin)

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200

CON

VDN VDN+Ghr (L) VDN+Ghr (H)

ET mRNA β-actin

150

* *# * # 100

0 CON

VDN

VDN+Ghr (L) VDN+Ghr (H)

Fig. 6. Expression of endothelin (ET) mRNA in rat aortas. CON, controls; VDN, rats given vitamin D3 and nicotine for 4 weeks; VDN + Ghr(L), calcified aortas treated with 30 nmol kg 1 day 1 ghrelin for 4 weeks; VDN + Ghr(H), calcified aortas treated with 300 nmol kg 1 day 1 ghrelin for 4 weeks. *P b 0.01 vs CON, #P b 0.01 vs CAL.

(Ang II) and urotensin II derived from cardiovascular tissues aggravated vascular calcification [3,20], while adrenomedullin, C-type natriuretic peptide and parathyroid hormonerelative peptide (PTHrP) attenuated this injury [3,21], which indicates that a disturbance in the vascular paracrine/ autocrine function might be an important inducer of calcification [11,20,21]. The stomach is the major source of circulating ghrelin, as determined by peptide quantification in the different regions of the gut and a 70% reduction in plasma levels after gastrectomy in rodents and humans [6]. Ghrelin is a strong gastrokinetic agent that links the endocrine control of energy balance and growth with the regulation of digestive function by activating its specific receptor (GHSR-1a) in the central tissues [5]. Binding sites specific for ghrelin and its mRNA expression exist in cardiovascular tissues such as ventricles, atria, aortas, coronary arteries, carotid arteries, endocardium and vena cava, which indicates that the cardiovascular system expresses the components of the ghrelin/GHSR axis, which may have an important autocrine/paracrine function in maintaining circulatory homeostasis [10,11]. Prolonged treatment with ghrelin markedly protects against cardiovascular damage in aged rats and rats with growth hormone deficiency and post-ischemic ventricular dysfunction [6]. Ghrelin also improves cardiac performance in rats with myocardial infarction, protects against diastolic dysfunction of myocardial stunning in isolated, perfused rabbit hearts and enhances left ventricular contractility in pigs with dilated cardiomyopathic features [22]. These results suggest that ghrelin is an endogenous factor with potent protective effects in cardiovascular diseases [6]. In our study, we established the rat model of vascular calcification by arterial calcium overload induced by excess doses of vitamin D3 and nicotine. Four weeks after treatment, aortic calcium content, 45Ca2+ deposition, ALP

activity and systolic blood pressure were significantly increased, but aortic OPN mRNA expression was decreased. Von Kossa staining showed calcium accumulation on the medial elastic fibers and positively stained black/brown areas and nodular structures in vascular media. These results are similar to those of Niederhoffer et al. [12]. In addition, we used rat VSMCs treated with h-glycerophosphate to induce calcification in vitro. h-glycerophosphate is a substrate for ALP; it could release inorganic phosphate to raise the local concentration of phosphorous, accelerate calcium and phosphorus deposition on cells and tissues, and induce the formation of calcified nodules [14]. As reported by Shioi et al. [14], we also observed that calcium content, 45 Ca2+ uptake and ALP activity were significantly increased in calcified VSMCs, and OPN mRNA expression was decreased. Von Kossa staining showed the formation of multicellular nodules. Compared with controls, calcified aortas showed significantly decreased plasma ghrelin levels and mRNA expression in aorta, which indicates that the local ghrelin system might be impaired during vascular calcification. After treatment with ghrelin, the aortic calcification was ameliorated, with both 30- and 300-nmol treatment, with decreased aortic calcium content, 45Ca2+ deposition and ALP activity, inhibited aortic calcium deposition and formation of nodular structures, and increased aortic OPN mRNA expression. Compared with 30-nmol ghrelin treatment, 300-nmol treatment was more potent in decreasing ALP activity in plasma and aortas. In calcified VSMCs in vitro, ghrelin (10 8~10 6 mol/L) decreased calcium deposition, ALP activity and calcium overload and increased OPN mRNA expression. These studies in vivo and in vitro reveal that ghrelin was downregulated in the circulation and aortas with vascular calcification, and the exogenous supplement of ghrelin could effectively antagonize vascular calcification, which indicates that endogenous ghrelin might be involved in the regulation of vascular calcification and that the local ghrelin system is a potential therapeutic target for diseases related to vascular calcification. Currently, little is known about how ghrelin is regulated when produced and secreted in local cardiovascular tissues [22]. In both rats and humans, the ghrelin gene, which locates at 3p26–p25 is made up of 4 exons and 3 introns, and the precursors contain 117 amino acids (preproghrelin). Kishimoto et al. [23] have cloned the human ghrelin gene and characterized the 5V-flanking region, from 2000 to 1 upstream from the translation start site. The gene contained a TATATAA element and putative binding sites for several transcription factors but not a typical GC or CAAT box. Functional analysis showed that promoter activity was increased by the deletion of nucleotides from 2000 to 605 but decreased by further deletions and that the TATATAA element was not functioning. Glucagon and its second messenger, cAMP, enhanced the promoter activity, which suggests that stimulated transcription of the ghrelin gene by glucagon might be responsible at least in part for

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increased ghrelin production during fasting. Our previous work [24] also showed that the plasma ghrelin level was increased in proportion to the severity of septic shock. Asakawa et al. [25] noted that ghrelin gene expression in rat stomachs was increased by fasting in ob/ob mice and was decreased by the administration of leptin and IL-1 beta. Peripherally administered ghrelin blocked IL-1 beta-induced anorexia and produced positive energy balance by promoting food intake and decreasing energy expenditure. The down-regulated mechanism of ghrelin observed in our work needs further study. Although ghrelin has multiple functions, including vasodilation, a positive isotropic role, decreased production of oxygen free radicals during myocardial ischemia and inhibition of inflammation and apoptosis, the mechanism involved in its cytoprotective effects is still unclear [6,9]. Ghrelin is an endogenous peptide ligand for GHSR, a typical G-protein-coupled 7transmembrane receptor [22]. In situ hybridization analyses revealed that the GHSR is widely expressed in the pituitary gland, hypothalamus, hippocampus and cardiovascular tissues [10]. Shimizu et al. [26] observed that the administration of ghrelin improves endothelial dysfunction and increases eNOS expression in rats through GH-independent mechanisms. Baldanzi et al. [27] reported that ghrelin inhibits apoptosis in primary cultured adult rat cardiomyocytes, H9c2 cells and endothelial cells induced by adriamycin in vitro through the activation of extracellular signalregulated kinase-1/2 and Akt serine kinases. Recently, Wiley and Davenport [7] identified ghrelin as a potent physiological antagonist of endothelin-1 by comparing endogenous vasodilators in the human internal mammary artery. Our previous work has shown that excess endothelin was involved in vascular calcification by inducing the overload of aortic calcium and transforming the VSMC phenotype to osteoblasts [20]. In this study, we observed that endothelin mRNA expression in aortas was upregulated, and endothelin content in plasma and aortas in calcified tissue was increased, although in calcified tissue treated with ghrelin, endothelin and its mRNA expression in plasma and aortas were all down-regulated. In addition, linear regression analysis revealed that ghrelin content in plasma was negatively correlated with endothelin content (r = 0.83, P b 0.01, n = 18). The results suggest that ghrelin’s action on vascular calcification might be in part through the interaction with endothelin and down-regulation of its mRNA expression and contents.

5. Summary Our results show that the expression and content of ghrelin are down-regulated in calcified rat aortas and VSMCs, but the expression and content of endothelin are up-regulated. Treatment with ghrelin can attenuate the calcification effectively, and down-regulate the expression and content of endothelin significantly. These results

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suggest that an impaired local ghrelin system might be involved in the process of vascular calcification. Ghrelin would be an important target in the prevention and therapy of vascular calcification, in part through its antagonism in the vascular endothelin system.

Acknowledgements This study was supported by the Major State Basic Research Development Program of The People’s Republic of China (G2000056905).

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